Proton translocation in corn coleoptiles: ATPase or redox chain?

A possible involvement of two different systems in proton translocation was investigated by simultaneous measurement of transmembrane electron flow and proton secretion in a pH-stat combined with a redoxstat. The pH gradient between cytoplasm and apoplast is probably maintained by an H+-pumping ATPase and by a second proton extrusion system, which seems to be linked to a redox chain with NAD(P)H as electron donor. Indole acetic acid inhibits both e-and H+ efflux, but only if the 'electron draw' from the outside is not too high. The electron draw depends on the hexacyanoferrate level at the plasmalemma surface and on the Ca2+ concentration. The inhibiting effect of auxin on e-and H+ efux in the presence of hexacyanoferrate can be only detected at low levels of bivalent cations and of the artificial electron acceptor. The inhibition of e-and H+ efflux by auxin requires high oxygen levels. idants like HCF III or HCI IV will very likely impair the functions of a variety of plasmalemma proteins, including channels, cotransporters, pumps, and auxin-receptors by oxidizing sulfhydryl groups of the proteins (6). In other words, a number of important transport functions may be impaired which would alter electron and/or proton movement. (c) Alcohols can be used to increase the level of intracellular NADH through the action of the cytoplasmic alcohol dehydrogenase (6, 10, 11). Alcohols, known to be substrates for alcohol dehydrogenase such as propan-1-ol, ethanol, and butan-1-ol increase net H+ efflux immediately; propan-2-ol and methanol have no effect. This stereoselectivity and the requirement of high 02 supply for alcohol action exclude an explanation as a membrane effect. Exogenous NADH was found to increase HCF III reduction of protoplasts (21) and of intact roots (29). Komor et al. (20) have demonstrated that reduction of externally applied NAD(P)H with concomitant acidificaton in the apoplast is without involvement of transmembrane steps. An independence of transplasma membrane proton gradient from NAD(P)H-HCF III oxidoredtction in maize root microsoms was described (24). We therefore avoided addition of NAD(P)H and tried to distinguish between H+-pumping ATPase and redoxchain-linked proton extrusion by the use of different 02 pressures as the ATP generating system and the plasmalemma NAD(P)H oxidase have very different Km values for 02.The aim of the present paper is to investigate the influence of hormones and other effectors on the proton extrusion and its possible linkage to transmembrane electron transfer. MATERIALS AND METHODS

Section: Methodsmentioning

confidence: 99%

Hormone Action on Transmembrane Electron and H⁺ Transport

Böttger¹,

Hilgendorf²

1988

“…H+ extrusion in the presence of ferricyanide might occur at the level of the redox chain, similarly to that known for the mitochondria and thylakoid membranes: then the plasmalemma redox chain would represent a mechanism of H+ extrusion alternate to the better known ATP-driven proton pump (14,23). A second possibility is that the reducing agent at the external plasma membrane surface is of the XH2/X type, which would donate electrons to Fe(CN)3-and release H+ into the medium (3,6). According to a third, alternative interpretation the electron transport to ferricyanide would activate the ATP driven pump, which would be the main mechanism responsible for H+ extrusion (17,27).…”

mentioning

confidence: 96%

“…The finding that ferricyanide reduction is associated with the acidification of the extracellular medium suggested a possible role of this system in ion transport across the plasma membrane, and in particular in H+ extrusion (6,11,12,14,18,29). H+ extrusion in the presence of ferricyanide might occur at the level of the redox chain, similarly to that known for the mitochondria and thylakoid membranes: then the plasmalemma redox chain would represent a mechanism of H+ extrusion alternate to the better known ATP-driven proton pump (14,23).…”

mentioning

confidence: 99%

Plasmalemma Redox Activity and H⁺ Extrusion

Marré

Moroni²,

Albergoni³

et al. 1988

Recent evidence demonstrates in plants the operation of a plasmalemma redox system transporting reducing equivalents from intracellular substrates to extracellular electron acceptors such as ferricyanide and ferric ion. The finding that ferricyanide reduction is associated with the acidification of the extracellular medium suggested a possible role of this system in ion transport across the plasma membrane, and in particular in H+ extrusion (6,11,12,14,18,29). H+ extrusion in the presence of ferricyanide might occur at the level of the redox chain, similarly to that known for the mitochondria and thylakoid membranes: then the plasmalemma redox chain would represent a mechanism of H+ extrusion alternate to the better known ATP-driven proton pump (14,23). A second possibility is that the reducing agent at the external plasma membrane surface is of the XH2/X type, which would donate electrons to Fe(CN)3-and release H+ into the medium (3, 6). According to a third, alternative interpretation the electron transport to ferricyanide would activate the ATP driven pump, which would be the main mechanism responsible for H+ extrusion (17,27).In an attempt to discriminate between these alternatives we assumed that they might result in a different behavior of the intracellular pH. The direct extrusion of H + by the redox system should not influence cytoplasmic pH, inasmuch as the two hydrogen atoms (2 electrons + 2 protons) coming from the oxidation of a respiratory substrate (XH2 to X) would be transported out of the cell: the two electrons would reduce two ferricyanide molecules and the two protons would be secreted as such into the extracellular medium. In contrast, the model coupling the electron efflux by the redox system with the extrusion of protons by the ATP-driven pump would implicate at least an initial acidification of the cytoplasm by the H + released in the cytosol during the transport of electrons to ferricyanide. This acidification would then be partially compensated by a secondary increase in activity of the ATP driven H+ pump. Thus, we measured the changes in intracellular pH (in cell sap, 'bulk cytoplasm' and vacuole; 1, 2, 22) associated with ferricyanide reduction and acidification of the external medium.The relative roles of the redox chain and of the H+ pump in ferricyanide-induced H + extrusion were investigated by influencing the activity of ATP-driven H + extrusion. As an activator of the ATP-drivep pump we used fusicoccin (recently shown to stimulate ATP-dependent electrogenic H+ transport in plasmalemma preparations; 24, 25). To inhibit the pump, we used erythrosine B and vanadate, two relatively specific inhibitors of the H+-ATPase of plasmalemma (10), both very active on H+ extrusion in vivo in Elodea leaves (19).As a plant material we chose Elodea densa leaves, very active in reducing ferricyanide (14) and also endowed with an efficient mechanism of electrogenic H + extrusion, presumably depending on the operation of the ATP-driven fusicoccin-stimulated and vanadate-inhibited H+ pump (1,19).All ...

“…Models of possible plasmalemma activities involving flavoprotein and this cytochrome have been discussed (10). There is recent evidence pointing to NADPH as the preferred electron donor for trans-plasmalemma redox functions (2,5,20,23), and to vanadate-insensitive proton pumping by the plasmalemma induced, but not energized, by blue light (1,3,15,22) …”

Section: Discussionmentioning

confidence: 99%

“…Considerable interest has been aroused by reports that NADH and NADPH can act as donors to unidentified electron carriers in or near the plasma membrane, often with an increase of 02 uptake (5,15,25) and enhanced ion transport (3,5,15). The properties of these systems are rather diverse; for instance, some investigators find cyanide sensitivity (16,25), other deny this (21); a pyridine nucleotide-dependent, SHAM2-stimulated oxy-' Supported by grants from CNR, and the Ministero Pubblica Istruzione (Rome), for years 1985 and 1986.…”

mentioning

confidence: 99%

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

Guerrini

Valenti²,

Pupillo³

1987

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis copsisted of two types of subunits, a (molecular weight 39,500) and b (37,0W) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a2b2) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hiu coefficients fN = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoproteip, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.The plasmalemma of plant cells contains electron transport chains able to transfer reducing equivalents from the cytoplasm to the outer surface. These chains are thought to operate in modifying the immediate plant environment, in cell wall growth and ionic balance (2,5,17). The best characterized function is transmembrane Fe"' reduction, effected by iron-stressed (23)